Anergic NKT cells are defective in transactivating B cells, conventional T cells, NK cells, and DCs. Naive mice or mice injected with α-GalCer (5 μg/mouse, i.p.) 1 month earlier were reinjected with α-GalCer or vehicle. (A) Expression of activation markers. Splenocytes were prepared after 24 hours and stained with different combinations of tetramer-PE, anti-B220–PerCP, anti–TCR-β–allophycocyanin, anti-NK1.1–PE, and/or anti-CD69–FITC. CD69 expression was evaluated on NK cells (B220^–TCR-β^–NK1.1^high), B cells (B220⁺TCR-β^–), and conventional T cells (B220^–TCR-β⁺tetramer^–). Alternatively, cells were stained with anti-CD11c–allophycocyanin, anti-CD80–FITC, and anti-CD86–PE. Expression of CD80 and CD86 was evaluated on CD11c^high DCs. (B) IFN-γ production by NK cells. Splenocytes were prepared at the indicated time points, cultured with GolgiPlug for 6 hours, and stained with anti-CD3–PerCP and anti-NK1.1–PE followed by anti–IFN-γ–FITC or isotype-control Ab. Intracellular IFN-γ expression by NK1.1^highCD3^– cells was evaluated. Specificity of staining was demonstrated with isotype controls (Supplemental Figure 1). (C) Graphical representation of the data from B. (D) Serum cytokine levels. Naive mice or mice injected 1 month earlier with α-GalCer were injected with α-GalCer or vehicle. Mice were bled at the indicated time points, and serum IL-4, IFN-γ, and IL-12 levels were measured by ELISA. Data shown represent the mean ± SEM of 4 mice per group. Representative data of 3 individual experiments are shown.